CN104784703A - Aptamer-based targeted delivery microRNA nanometer carrier as well as preparation method and application thereof - Google Patents
Aptamer-based targeted delivery microRNA nanometer carrier as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an aptamer-based targeted delivery microRNA nanometer carrier as well as a preparation method and application thereof. A targeting molecule is a sulfydryl-modified MUC-1molecule, and can be combined with cancer-inhibiting microRNA-34a, so that a MicroRNA-nanodiamond-protamine complex (MNPs) becomes AMNPs with an active targeting function; through electrostatic interaction, a negatively-charged aptamer-microRNA (Apt-miR) chimera is adsorbed to a cationic protamine-nanodiamond carrier to form a trinary compound carrier. The therapeutic effect of a nanotransport system on a non-small cell lung cancer is researched; the Apt-miR chimera is low in immunogenicity; the modified MUC-1 aptamer has high chemical stability in vivo, and can be chemically synthesized easily; in order to improve the transfection efficiency, a protamine-modified nanodiamond material (NPs) is used for helping miRNA to increase accumulation in a tumor part through permeability and retention enhancing effects.
Description
Technical field:
The invention belongs to nano material field of biology.The present invention relates to nano-carrier of a kind of load microRNA of tool targeting and preparation method thereof, be specifically related to the preparation method of Nano diamond carrier and the application in lung cancer therapy thereof of the modification of a kind of MUC-1 targeting protamine.
Background technology:
MicroRNA (miRNA) is a class endogenous non-coding RNA, and length is between 20-25 base.MicroRNA, by combining with the 3 '-noncoding region (3 '-UTR) of mRNA, forms local base pairing to suppress the translation of mRNA or to make it degrade.A kind of microRNA can be combined with multiple sites of mRNA, a kind of expression of results of gene also may regulate by different microRNA.Many microRNA abnormal expression in various cancers, the generation of prompting microRNA and cancer develops closely related.Different MicroRNA plays different roles in cancer, some microRNA are had to have the effect of similar oncogene, higher at certain cancers cells, can by negative regulation tumor suppressor gene or by cell cycle regulation, differentiation, apoptotic process, stimulate cellular proliferation, promote inside tumor angiogenesis, these carcinogenecity microRNA are called as oncomirs; Another kind of microRNA then plays the role of strong antioncogene, is referred to as and presses down cancer miRNA.
Increasing miRNA is applied in treatment of cancer, but still there is important problem, is exactly how to ensure that these miRNA without gene specific enter the efficiency that tumor cell plays therapeutic effect.Therefore, for the outstanding stable carrier with targeting effect of miRNA searching is the shortcut playing miRNA curative effect better.Current Therapeutic cancer is the most frequently used and effective method is still combined chemotherapy radiotherapy, but this method inevitably damages human body.Therefore, it is possible to the novel carriers sending specific antitumor drug is more and more favored.These pharmaceutical carriers can make up the limitation of medicine, control medicine rate of release in vivo, and can reduce medicine to the work of non-tumor cell in order to avoid Cytotoxic generation.Nano diamond is a kind of carbon nanomaterial, and use Nano diamond as microRNA vehicle treatment cancer, have toxicity low, biocompatibility is high, can extend by the stagnation duration etc. of medicine carrying thing at tumor locus.
Aptamer (Aptamer) is single stranded DNA or the RNA that a class has specific binding target molecules, nucleic acid or protein.Nano material carrier can be combined with aptamer further after chemical modification, targeted therapy efficiency is improved widely.Aptamer through nano-carrier load greatly reduces its immunogenicity, not easily by organism degrades, improves the enriching of own load medicine or molecule, strengthens therapeutic effect.
Summary of the invention:
The present invention is nano-carrier preparation method and the application of a kind of aptamers targeted delivery microRNA.Specifically, the Nano diamond carrier that the present invention modifies with protamine, using aptamer-microRNA chimera (Apt-miR) as functional loaded article targeted therapy of lung cancer, this complex carrier (aptamer-microRNA-Nano diamond) is prepared efficient and convenient, cytotoxicity is low, and targeted therapy is respond well.
A nano-carrier of the load microRNA of tool targeting, is characterized in that, described carrier is the Nano diamond modified through Protamine sulfates., and this carrier is cationic.
Further, wherein Nano diamond is produced by explosion method.
Further, Protamine sulfates. derives from salmon.
Further, wherein single Nano diamond particle diameter is 4-6nm, and average kinetic is of a size of 40-60nm, and surface zeta potential current potential is 13-18mV.
Further, wherein said microRNA is Hsa-miR-34a.
Further, targeting molecule is the MUC-1 molecule of sulfydryl modification.
Prepare the method for Nano diamond-MUC-1-miR-34a chimera composite Nano carrier A MNPs, it is characterized in that comprising the following steps:
(1) the Nano diamond gel solution of 150mg/mL is dissolved in deionized water, dilutes for final concentration 1mg/mL; ND solution after the ultrasonic reaction that spends the night under 100W power dilution;
(2) 50mg Protamine sulfates. is dissolved in 5mL deionized water, makes Protamine sulfates. solution final concentration be 10mg/mL;
(3) by Nano diamond gel solution and Protamine sulfates. solution in mass ratio 1:15 be mixed into NPs solution, hatch for 37 DEG C and carry out self-assembling reaction in 48 hours;
After (4) 48 hours, by centrifugal for NPs mixed liquor 12000rpm 15 minutes; Abandon supernatant, the NPs bead obtained uses deionized water again to disperse, and recentrifuge washing of precipitate repeats this step 3 time; Finally precipitation lyophilizing is dissolved in deionized water, adjustment concentration is 0.6mg/mL;
(5) the miR-34a mimics that SPDP modifies is dissolved in 1mL without in RNA enzyme water, obtain the solution that concentration is 80 μ g/mL RNA, mix with NPs solution according to the solution of mass ratio 1:4RNA, incubated at room makes it carry out assembling for 30 minutes and is obtained by reacting MNPs;
(6) in MUC-1:miR-34a mol ratio be the ratio of 1:2 by the MUC-1 molecule of sulfydryl modification and MNPs night incubation, be assembled into AMNPs.
Further, wherein the miR-34a mimics that SPDP modifies is specially by step (5): 1.25nmol5 ' end band had amino miR-34a mimics to be dissolved in 200 μ L PBS and obtain miR-34a diluent, be that 0.0625nmol SPDP cross-linking agent is dissolved in 20 μ L DMSO and obtains SPDP working solution by 1:20 by SPDP:miR-34a mol ratio simultaneously, miR-34a diluent is mixed with SPDP working solution, hatches 2 hours.After reaction, excessive SPDP by ultra-filtration centrifuge tube repeatedly centrifugalize go out.
AMNPs prepared by said method.
AMNPs prepared by said method is as the application in lung cancer therapy medicine.
First, the present invention uses through Protamine sulfates. (Protamine sulfate, PS) the Nano diamond carbon nanomaterial (Nanodiamond modified, ND) as the delivery vehicles (NPs) of microRNA, its advantage is that cell transfecting efficiency is high, can quickly and efficiently by Cell uptake.Secondly, utilize Mucin-1 (MUC-1) aptamer as target ligand, design and synthesis one segment length is about the Deoxydization nucleotide single-stranded structure of 25base, chimeric construct is formed with microRNA-34a (miR-34a), be carried on NPs, to increase the targeting that it is delivered to pulmonary carcinoma, thus strengthen the therapeutic efficiency of microRNA.
Protamine is a kind of alkaline protein being mainly derived from Fish, has higher heat stability, shows antagonistic property and biological activity simultaneously.Water miscible Protamine sulfates., by hydrogen bond, surface charge effect, can be adsorbed in Nano diamond surface; Nano diamond is a kind of carbon nanomaterial, compared with other nano material, there is toxicity little, the features such as biocompatibility is better, the a large amount of hydroxyl of its surface enrichment, carboxyl, the carbonyl amino easily in protamine is combined, form the nano-particle of protamine parcel Nano diamond, after this modifies, the microRNA with negative electricity, with positive charge, can be adsorbed in NPs surface.
Mucin1 mucin (MUC-1) is a kind of cell surface glycoprotein, belongs to I type transmembrane protein, is expressed in the epithelial cell of each organ-tissue more under normal circumstances.It is all express higher than normal level that this glycoprotein comprises non-small cell lung cancer cell surface in the tumor of numerous species.Because MUC-1 aptamer and miR-34a are nucleotide, therefore chimera can be formed after SPDP cross-linking agent is modified; SPDP (3-(2-pyridine dimercapto) propanoic acid N-hydroxy-succinamide ester) can react with the amino on compound and generate stable amido link, α-pyridine radicals simultaneously in molecule is responsive to sulfydryl, sulfydryl-disulfide group exchange reaction occurs and generates new disulfide bond.These two kinds reactions are carried out simultaneously and are not interfere with each other under suitable condition.This cross-linking method side reaction is few, and conjugate productive rate is high.
Due to nano-diamond particles cluster existence in aqueous, so need to be disperseed by supersonic vibration before using.But nano-diamond particles still can not be divided into independent part by this dispersion, be still and exist with tuftlet pattern, characterizing its surface zeta potential current potential known through ZETA current potential is about 15mV, and under steady statue, diameter is about about 52nm, and single nano-particle theoretical diameter is about 4nm.The Nano diamond of agglomerating existence can increase the load capacity of microRNA, and this is also use Nano diamond as one of advantage of carrier.
Accompanying drawing explanation
Fig. 1 is NPs, MNPs, AMNPs self assembly illustraton of model;
Fig. 2 is the surface zeta potential current potential of MNPs and AMNPs complex;
Fig. 3 A is one of agarose-formaldehyde degeneration gel electrophoresis figure.
Fig. 3 B is agarose-formaldehyde degeneration gel electrophoresis figure bis-.
Wherein No. 1 swimming lane is MUC-1Aptamer, and No. 2 swimming lanes are miR-34a, and No. 3 swimming lanes are the mixture of MUC-1 and miR-34a, and No. 4 swimming lanes are MUC-1-miR-34a chimera;
Fig. 4 observes AMNPs and MNPs by the ability of A549 cellular uptake under laser confocal microscope.Wherein miR-34a is by red fluorescence dyestuff Cy-3 labelling, and red light intensity is directly proportional to the amount of miR-34a;
Fig. 5 is after difference transfection miR-34a mimics (Naked miR-34a), NPs, MNPs and AMNPs, the expression of miR-34a in A549 cell;
Fig. 6 is that Transwell basis of microscopic observation result and histogram results display miR-34a can suppress A549 cell migration;
Fig. 7 is that flow cytomery result display miR-34a can induce non-small cell lung cancer cell apoptosis.
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The preparation of embodiment 1 Nano diamond working solution
The Nano diamond used in the present invention is gel solution (15%w/v), and purchased from NanoCarbon Research Institute Ltd. (catalog number: 386-8567), particle diameter is 3.6 ± 0.7nm.
Before use, Nano diamond (Nanodiamond, the ND) gel solution of 150mg/mL is dissolved in deionized water, dilutes for final concentration 1mg/mL.ND solution after the ultrasonic reaction that spends the night under 100W power dilution.
Embodiment 2 Protamine sulfates. decorated nanometer diamond (NPs)
The Protamine sulfates. used in the present invention is purchased from Sigma Adrich company (catalog number: PS4020).
50mg Protamine sulfates. (Protamine sulfate, PS) is dissolved in 5mL deionized water, makes Protamine sulfates. solution final concentration be 10mg/mL.By Nano diamond gel solution and Protamine sulfates. solution in mass ratio 1:15 be mixed into NPs solution, hatch for 37 DEG C and carry out self-assembling reaction in 48 hours.
After 48 hours, by centrifugal for mixed liquor 12000rpm 15 minutes.Abandon supernatant, the NPs bead obtained uses deionized water again to disperse, and recentrifuge washing of precipitate repeats this step 3 time.Finally precipitation lyophilizing is dissolved in deionized water, adjustment concentration is 0.6mg/mL.
MiR-34a modified by embodiment 3 SPDP cross-linking agent
The miR-34a mimics used in the present invention is synthesized by Guangzhou Rui Bo biotech company, and its sequence is, positive-sense strand: 5 '-UUCUCCGAACGUGUCACGUdTdT-3 '; Antisense strand: 5 '-ACGUGACACGUUCGGAGAAdTdT-3 ', the Nsuccinimidyl-3-used in the present invention (2-pyridyldithio) propionate (SPDP) is purchased from Thermo-Fisher Scientific company (catalog number: 21857).
SPDP cross-linker cross-linking agent is dissolved in DMSO by molar concentration 1:20 hatches 2 hours, 1.25nmol 5 ' end band is had amino miR-34a mimics to be dissolved in 200 μ L PBS simultaneously.After reaction, excessive SPDP make membrane ultrafiltration centrifuge tube repeatedly centrifugalize go out.
The preparation of embodiment 4 MiR-34a-NPs (MNPs)
The miR-34a mimics that SPDP modifies is dissolved in 1mL without in RNA enzyme water, and obtain the solution that concentration is 80 μ g/mL RNA, mix according to mass ratio 1:4 with PNs solution, incubated at room makes it carry out assembling for 30 minutes and is obtained by reacting MNPs.
The preparation of embodiment 5 Aptamer-miR-34a-NPs (AMNPs)
MUC-1Aptamer-MNPs conjugated body (AMNPs) is formed in order to mucoprotein MUC-1 (Mucin-1) aptamers (MUC-1Aptamer) be connected on MNPs, MUC-1 sulfhydrylation is modified by we, then form miR-34a-aptamer chimera in miR-34a:MUC-1 with the ratio of 2:1 and MNPs night incubation, be assembled into AMNPs.
Embodiment 6 NPs, the detection of MNPs and AMNPs particle diameter, surface potential
By the NPs of synthesis, MNPs and AMNPs is diluted to the concentration of 60 μ g/ml respectively after hatching 15min, by particle diameter and the surface zeta potential current potential of Zetasizer Nano ZS (Malvern, Worcestershire, U.K.) test compound thing.The parameter of test particle diameter is 25 DEG C, 90 ° of angle of scatterings.The mensuration of surface potential is based on nanoparticle electrophoretic mobility in the solution, measures in automatic mode.
As shown in Figure 1, wherein, the potential measurement result of MNPs, AMNPs as shown in Figure 2 for NPs, MNPs, AMNPs illustraton of model NPs that self assembly is formed.Can find out, on the basis of MNPs, loaded article is replaced by miR-34a-MUC-1 chimera by miR-34a, after being assembled into AMNPs, its complex particle diameter is increased to 190.1nm (± 3nm) by 186.7nm (± 5nm); Surface zeta potential current potential changes into-25.6mV (± 2.3mV) by-20.7mV (± 1.2mV).This result can prove successfully to be connected in MNPs system with the MUC-1Aptamer of negative electricity well.
The chimeric connection of embodiment 7 detected through gel electrophoresis Aptamer-miR-34a
Prepare agarose-formaldehyde degeneration glue (2%, 100ml), weigh 3g low melting-point agarose powder dissolution in appropriate DEPC water, boil until dissolve completely, then add appropriate DEPC water and be settled to 93ml.After solution is cooled to about 60 DEG C, add 30ml 5 × MOPS-EDTA Buffer and 27ml 37% formaldehyde (operating in fume hood), leave standstill 1 hour in room temperature after glue.
Get 15 μ l miR-34a mimics, MUC-1Aptamer and AMPNs mixes with 5 μ l RNA loading buffer (+EB), under 100V (constant voltage) condition, electrophoresis to bromophenol blue migrates to 2/3 right position of glue, uses Alpha Innotech gel imaging system observed result and takes pictures.
Electrophoresis result is as shown in Fig. 3, A, and agarose gel electrophoresis figure represents that left several Article 1 miR-34a band can clearly be presented on agarose gel; And Article 2 MNPs band, due to miR-34a adsorb by NPs, its overall charge is weakened, and therefore cannot move on agarose gel; Article 3 MNPs+SDS band represents that SDS destroys the structure of MNPs, miR-34a is spun off and demonstrates electrophoretic band after MNPs and SDS mixing.In Fig. 3, B 1,2,3, No. 4 bands represent MUC-1Aptamer respectively, the mixture of miR-34a, MUC-1 and miR-34a, MUC-1-miR-34a chimera, as can be seen from electrophoresis result, by the method in the present invention, MUC-1 and miR-34a can form chimeric construct well.
Embodiment 8 AMPNs absorbs the inspection of ability
1, cell culture and inoculation
The present invention uses Lines A549 as cell experiment object, and A549 is purchased from purchased from Chinese Academy of Sciences's cell bank.Be resuspended in 5mL PBS solution after centrifugal for the cell suspension of acquisition, after using blood counting chamber counting, get in the DMEM culture medium that 1 × 105 cell inserts containing 15mL preheating.Culture medium be mixed in advance 10% hyclone and 1% streptomycin-penicillin dual anti-, carry out in whole operation and aseptic operating platform.Culture medium is inserted aseptic incubator, 37 DEG C, cultivate under 5%CO2 condition, to go down to posterity to cell confluency to 90% or frozen.
2, the transfection of material
Use the AMNPs transfectional cell of new preparation, prepare matched group MNPs simultaneously, AMNPs, MNPs and the Opti-MEM culture medium prepared is mixed in proportion, makes the final concentration of miR-34a be 100nM.Culture fluid in removing culture dish, use PBS to clean cell 3 times, add the mixed liquor 1mL that above-mentioned dilution is good, undressed A549 cell as a control group, hatches 6 hours for 37 DEG C.After 6 hours, remove the liquid in each culture dish, add the DMEM culture medium containing 10%FBS of preheating 37 DEG C, 37 DEG C are continued cultivation 18 hours.
3, Laser Scanning Confocal Microscope detects
The cell that transfection is good can use confocal laser scanning microscope through fixing, dyeing after processing.Culture medium in removing culture dish, adds 1mL 4% paraformaldehyde fixative and is fixed 30 minutes.Then in the cell fixed, add 1mL Triton X-100 (Triton X-100), with dissolved cell film surface lipids, make further dye enter cell.Incubated at room, after 10 minutes, adds 5% (w/v) BSA confining liquid that 1mL prepares in advance, continues at room temperature to close 1 hour.Finally add 500 μ L DAPI dyeing liquors, room temperature lucifuge is hatched and is dyeed for 3 minutes.Removing dyeing liquor, uses PBS to clean culture dish 5 times to remove the interference of too much dyeing liquor to the visual field.Confocal laser scanning microscope cell: arranging excitation wavelength is that 570nm, emission wavelength 660nm observe red Cy-3 fluorescent labeling.
As shown in Figure 4, in AMNPs group, Cy-3 fluorescence intensity is far above MNPs group for result, and the targeting due to MUC-1 is described, make whole nano-carrier system more efficiently absorb by A549 cell, its Targeting Effect is obvious.Can see in enlarged drawing, the miR-34a of Cy-3 labelling can enter cell and be enriched in nucleus (blueness) around.
Embodiment 9 Real-time PCR detects the expression of miR-34a
1, prepare before detecting
According to the method in embodiment 8, use the AMNPs transfectional cell of new preparation, prepare matched group MNPs simultaneously.Then use Trizol method to extract the total serum IgE of each group of cell, and use NCode
tMits reverse transcription is cDNA by VILOTM miRNA cDNA Synthesis (catalog number MIRC-50, Life Technologies) test kit, prepares the expression detecting wherein microRNA.
2, RT-PCR detects the expression of miR-34a
The present invention uses Ncode
tMeXPRESS
greenER
tMmiRNA qRT-PCR test kit (catalog number A11193-051, Life Technologies) detects the expression of miR-34a.First design Forward primer is needed, specific design principle reference reagent box service manual.
As shown in Figure 5, after MNPs and AMNPs transfection A549 cell, its miR-34a expression is higher than matched group for RT-PCR result.In addition, due to the targeting of AMNPs, in AMNPs transfection group, the expression of miR-34a has obviously upper ascending effect.
Embodiment 10 Transwell tests and detects A549 cell transfer ability
According to the method in embodiment 8, use the AMNPs transfectional cell of new preparation, prepare matched group MNPs simultaneously.Growth of Cells to be transfected merge to 60% to 70% time be replaced by plasma-free DMEM medium hunger and cultivate 24 hours.After 24 hours, peptic cell is also replaced by serum-free medium, and adjustment cell quantity is 1 × 10
5individual/100 μ L, by cell suspension piping and druming evenly, add 100 μ L cell suspension in each Transwell cell, cell periphery adds DMEM culture medium 500 μ L containing 10%FBS as inducer, and often group establishes 3 repeating holes.Cultivate after 8 hours, use 4% paraformaldehyde fixed cell, and use 0.2% crystal violet solution color.Cell to be immersed in clean PBS and in basis of microscopic observation, for each cell, to get 8 random field under microscope and take pictures and count.
Result as shown in Figure 6, A be the normal A549 cell cultivated in contrast; B is the A549 cell of NPs transfection; C is the A549 cell of MNPs transfection; D is the A549 cell of AMNPs transfection; E is the cell relative migration ability that rectangular histogram represents.Can find out, MNPs and AMNPs inhibits the migration of cell to some extent after miR-34a is transported to A549 cell, but AMNPs group demonstrates more significantly cellular migration inhibition ability.
Embodiment 11 Annexin V-PI detects apoptosis
Whether have to detect MiR-34a the ability reducing the resistance to apoptosis of A549 cell, this research uses the apoptosis situation of the two dye method dyeing of flow cytomery Annexin V-FITC/PI (propidium iodide).Propidium iodide is a kind of double-stranded DNA fluorescent dyestuff, and fluorescence intensity is directly proportional to the content of double-stranded DNA.Nucleus by the cell strengthened through apoptosis middle and advanced stage permeability of cell membrane, can be dyed redness by propidium iodide.Annexin V is a kind of cardiolipin binding protein, and cell membrane that can be early stage with apoptosis by the Phosphatidylserine exposed outside cell is combined.Therefore Annexin V and PI is dyeed in two positive (Annexin V+/PI+) with the use of non-viable apoptotic cell can be combined by FITC and PI with non-viable non-apoptotic cell simultaneously.
As shown in Figure 7, miR-34a can cause A549 apoptosis to result.A be the normal A549 cell cultivated in contrast; B is the A549 cell of NPs transfection; C is the A549 cell of MNPs transfection; D is the A549 cell of AMNPs transfection; E is the percentage of cerebral apoptosis that rectangular histogram represents.Result display AMNPs group can obvious trigger cell early apoptosis.
Claims (10)
1. a nano-carrier of the load microRNA of tool targeting, is characterized in that, described carrier is the Nano diamond modified through Protamine sulfates., and this carrier is cationic.
2. carrier according to claim 1, wherein Nano diamond is produced by explosion method.
3. carrier according to claim 1, Protamine sulfates. derives from salmon.
4. carrier according to claim 1, wherein single Nano diamond particle diameter is 4-6nm, and average kinetic is of a size of 40-60nm, and surface zeta potential current potential is 13-18mV.
5. the nano-carrier of the load microRNA of tool targeting according to claim 1, wherein said microRNA is Hsa-miR-34a.
6. microRNA targeting delivery vehicles according to claim 1, targeting molecule is the MUC-1 molecule of sulfydryl modification.
7. prepare the method for Nano diamond-MUC-1-miR-34a chimera composite Nano carrier A MNPs, it is characterized in that comprising the following steps:
(1) the Nano diamond gel solution of 150mg/mL is dissolved in deionized water, dilutes for final concentration 1mg/mL; ND solution after the ultrasonic reaction that spends the night under 100W power dilution;
(2) 50mg Protamine sulfates. is dissolved in 5mL deionized water, makes Protamine sulfates. solution final concentration be 10mg/mL;
(3) by Nano diamond gel solution and Protamine sulfates. solution in mass ratio 1:15 be mixed into NPs solution, hatch for 37 DEG C and carry out self-assembling reaction in 48 hours;
After (4) 48 hours, by centrifugal for NPs mixed liquor 12000rpm 15 minutes; Abandon supernatant, the NPs bead obtained uses deionized water again to disperse, and recentrifuge washing of precipitate repeats this step 3 time; Finally precipitation lyophilizing is dissolved in deionized water, adjustment concentration is 0.6mg/mL;
(5) the miR-34a mimics that SPDP modifies is dissolved in 1mL without in RNA enzyme water, obtain the solution that concentration is 80 μ g/mL RNA, mix with NPs solution according to the solution of mass ratio 1:4RNA, incubated at room makes it carry out assembling for 30 minutes and is obtained by reacting MNPs;
(6) in MUC-1:miR-34a mol ratio be the ratio of 1:2 by the MUC-1 molecule of sulfydryl modification and MNPs night incubation, be assembled into AMNPs.
8. method as claimed in claim 7, wherein the miR-34amimics that SPDP modifies is specially by step (5): 1.25nmol 5 ' end band had amino miR-34a mimics to be dissolved in 200 μ LPBS and obtain miR-34a diluent, be that 0.0625nmol SPDP cross-linking agent is dissolved in 20 μ L DMSO and obtains SPDP working solution by 1:20 by SPDP:miR-34a mol ratio simultaneously, miR-34a diluent is mixed with SPDP working solution, hatches 2 hours.After reaction, excessive SPDP by ultra-filtration centrifuge tube repeatedly centrifugalize go out.
9. the as claimed in claim 7 or 8 AMNPs for preparing of method.
10. as claimed in claim 7 or 8 the AMNPs for preparing of method as the application in lung cancer therapy medicine.
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| WO2018082126A1 (en) * | 2016-11-02 | 2018-05-11 | 四川大学 | Drug loading system made of tdns-as1411-nucleic acid drug composite nanometer material, and preparation method therefor |
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| CN108753770A (en) * | 2018-06-01 | 2018-11-06 | 临沂大学 | A kind of gene nano probe and its preparation method and application for lung cancer-targeted treatment |
| CN109276558A (en) * | 2018-09-19 | 2019-01-29 | 北京工业大学 | Functionalized nano diamond drug-loading system and preparation method with targeting |
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| CN110917121B (en) * | 2019-12-11 | 2021-01-05 | 四川大学 | APD hybrid nano system and construction method and application thereof |
| CN112485452A (en) * | 2020-12-08 | 2021-03-12 | 北京工业大学 | Method for quantifying protein abundance by using metal clusters as artificial antibodies |
| CN112485452B (en) * | 2020-12-08 | 2023-10-10 | 北京工业大学 | Method for quantifying protein abundance by using metal cluster as artificial antibody |
| CN113181374A (en) * | 2021-05-08 | 2021-07-30 | 中南大学 | Spherical microRNA and preparation method and application thereof |
| CN113181374B (en) * | 2021-05-08 | 2022-05-13 | 中南大学 | Spherical microRNA and preparation method and application thereof |
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